Clinical and genomic features of a Listeria monocytogenes fatal case of meningitis in Madagascar

Abstract Listeriosis constitutes a significant public health threat due to its high mortality rate. This study investigates the microbiological and genomic characteristics of Listeria monocytogenes isolates in Madagascar, where listeriosis is a notifiable disease. The analysis focuses on a fatal case of meningeal listeriosis in a 12-year-old child. Genomic analysis revealed a novel cgMLST type (L2-SL8-ST8-CT11697; CC8, serogroup Iia) with typical virulence and antibiotic resistance profiles. These isolates, unique to Madagascar, formed an independent clade in the phylogenetic tree. This study presents the first genomic characterization of Listeria isolates in Madagascar, highlighting the necessity of ongoing genomic surveillance to strengthen listeriosis prevention and control strategies in the region.


Impact Statement
This study's significant findings on Listeria monocytogenes infections in Madagascar highlight the elevated risk among specific populations, particularly pregnant women from sub-Saharan Africa and the Maghreb region.The research reveals previously undocumented manifestations of the disease, expanding our understanding of its clinical presentation.Crucially, genomic analysis has identified a novel strain unique to Madagascar, providing valuable insights for tracing the pathogen's origins and transmission.The presence of specific virulence genes and antibiotic resistance profiles underscores the need for tailored prevention strategies.This pioneering genomic characterization lays the groundwork for targeted interventions and enhances public health initiatives, ultimately contributing to the prevention of listeriosis in the region.

INTRODUCTION
Listeriosis is one of the most serious food-borne illnesses, caused by the Gram-positive bacteria Listeria monocytogenes.It is a relatively rare disease, with 0.1 to 10 cases per million inhabitants per year, depending on the country and region.Although the number of listeriosis cases is low, the high mortality rate associated with this infection makes it a worrying public health issue [1].In pregnant women, listeriosis is higher in populations from sub-Saharan Africa and the Maghreb than in those from Latin America and the Caribbean.Among the 107 cases of maternal-neonatal listeriosis, 35 (33 %) originated from the Maghreb or sub-Saharan Africa, a rate three times higher than that observed in the general pregnant population in France in 2010, as indicated by national records (88 000 [11 %] out of 832 000 women, P<0.0001) [2].
Listeriosis is a notifiable disease in Madagascar.A recent study [3] documented the case of a 75-year-old man with an aortic bioprosthesis, who was admitted with polyarthritis in an afebrile state.Blood cultures confirmed the presence of L. monocytogenes, leading to the diagnosis of Listeria endocarditis and spondylodiscitis, both uncommon manifestations of listeriosis.The patient showed improvement following antibiotic therapy [3].Here we describe a case of invasive listeriosis at the CHU Befelatanana in Madagascar.

CASE PRESENTATION
A 12-year-old patient was admitted for meningeal syndrome, with a history of hyperthermic convulsive seizure at age 3. The patient is the first child in a sibling group of three, with an up-to-date immunization schedule.There was no similar case in the family.We received a sample of the cerebrospinal fluid and blood cultures.Direct examination of the CSF revealed hyperleukocytosis with a predominance of polynuclear neutrophils, hyperproteinorrachia, and hypoglycorrachia.Solid and liquid cultures were positive for a Gram-positive bacillus, which was identified as L. monocytogenes using the BioFire FilmArray Meningitis/Encephalitis (ME) Panel (Biomérieux, Marcy-l'Étoile, France).Concurrently, the blood culture also grew a Gram-positive bacillus with a 48 h growth period.Unfortunately, despite appropriate treatment according to the Common Infections Guidelines in Madagascar, 2019 edition [4] with ceftriaxone, gentamicin, and vancomycin, the patient did not survive.The antimicrobial susceptibility testing (AST) is not performed at the time of patient admission due to cost constraints for patients, especially considering the urgency of promptly treating bacterial meningitis, particularly in children.We used methods such as Gram-staining and Matrix-assisted laser desorption/ionization -time of flight mass spectrometry (MALDI-TOF MS) using the MALDI-TOF Biotyper System with the BDAL spectra database containing 8 468 MSPs, and the IVD containing 8 326 MSPs, for bacterial identification and the extraction method with 70 % formic acid and acetonitrile.The results indicated the presence of Gram-positive bacteria (Fig. 1), with the identification as L. monocytogenes with a score of >2.0 (Table 1).We notified the appropriate authorities; however, as of our current understanding, the origin remains undetermined.
Isolate characterization was performed by whole genome sequencing with Illumina technology and genomic analysis with TORMES pipeline [5] and VirulenceFinder [6,7].PCR-serogrouping, multilocus sequencing typing (MLST), core genome MLST (cgMLST) and virulence and resistance profiles were performed using BIGSdb-Listeria [6,[8][9][10][11][12][13][14].We conducted a phylogenetic analysis using core genome alignments that were not purged for genetic recombination, obtained with Parsnp 1. Identification results on the MALDI-TOF MS for the two strains of Listeria monocytogenes.If the score is between: 2.00-3.00, the identification is performed with a high confidence level; 1.70-1.99,the identification is performed with a low confidence level; 0.00-1.69,no identification is attributed to the organism  [15], incorporating publicly available strains from the BIGSdb-Listeria database belonging to clonal complex CC8 (as of 9 September 2023).
This new type differs by 12 allelic differences from L2-SL8-ST8-CT750, which has been found in Europe (Denmark, Poland, Estonia) and Latin America (Chile).Our phylogenetic analysis (Fig. 2) confirms that the Malagasy isolates exhibit a high degree of genetic similarity and form an independent clade, distinct from those originating in other countries.

DISCUSSION
To the best of our knowledge, this work represents the first genomic characterization of Listeria isolates from Madagascar.
Further sequencing efforts will help to identify sources of contamination and improve listeriosis surveillance and prevention efforts in our region.The discovery of a novel strain specific to Madagascar highlights the uniqueness of Listeria circulating in the local population.This groundbreaking research sets the stage for further sequencing efforts, aiming to identify contamination sources and enhance listeriosis surveillance and prevention strategies within Madagascar.The findings underscore the importance of integrating genomic data into public health initiatives, enabling targeted interventions, swift outbreak responses, and tailored patient care.Ultimately, this study serves as a crucial foundation for comprehensive research and collaborative efforts to mitigate the impact of listeriosis in Madagascar.

Fig. 2 .
Fig. 2. Phylogenetic tree based on the core genome alignment of Malagasy isolates and 100 publicly available CC8 sublineage SL8 genomes from BIGSdb-Listeria (as of 9 September 2023).From the innermost to the outermost circle, the first circle represents the distribution by ST, the second represents the distribution by continent, and the last one represents the isolation sources (b) with a zoom on the clades containing strains from Madagascar (a).